The present invention pertains to compositions related to proteins which function in controlling biology and physiology of mammalian cells, e.g., cells of a mammalian immune system. In particular, it provides purified genes, proteins, antibodies, and related reagents useful, e.g., to regulate activation, development, differentiation, and function of various cell types, including hematopoietic cells.
AK155 is a cytokine that is structurally related to interleukin-10 (IL-10). A number of cytokines have been classed as IL-10-like, based on their structure. The IL-10-like cytokines include IL-20, IL-22, and mda-7, as well as AK155 (Moore, et al., (2001) Annu. Rev. Immunol. 19:683-765; Moore, et al. (1993) Annu. Rev. Immunol. 11:165-190). Cytokines are signaling molecules that mediate communication between cells, e.g., between cells of the immune system. Once secreted, cytokines travel to a different or identical cell, bind to a membrane-bound receptor, and provoke a series of events such as protein phosphorylation or gene activation, where these events result in changes in phagocytic or secretory activity, or changes in migration, differentiation state, and proliferation activity.
The cytokines may be classed into those associated with increased inflammation (Th1-type response) and decreased inflammation (Th2-type response). The Th1-type response is characterized by increases in production of interferon-xcex3 (IFN-xcex3), tumor necrosis factor-xcex1 (TNF-xcex1), TNF-xcex2, IL-12, IL-18, and other pro-inflammatory cytokines (Sallusto, et al. (1998) Immunol. Today 19:568-574). IL-17 is generally a pro-inflammatory cytokine (Fort, et al. (2001) J. Immunol. 15:985-995). Th2-type response is characterized by increases in production of cytokines that suppress inflammation, e.g., IL-4, IL-5, IL-10, and IL-13 (Liu, et al. (2001) Nature Immunol. 2:585-589). IL-25 appears to suppress inflammation (Fort, et a. (2001) J. Immunol. 15:985-995).
Within one interleukin family, such as the IL-1 family, different interleukins may be functionally related to each other in that they influence the course of one event, but exert opposite effects on that event. For example, 1L-1xcex5 has a pro-inflammatory effect, while IL-1xcex4 has an anti-inflammatory effect (Debets, et al. (2001) J. Immunol. 167:1440-1446). IL-17 and IL-25 are examples of two other cytokines which are structurally similar, but produce opposite biological effects. The structure of any particular cytokine therefore serves as a guide to determining its physiological function.
An analysis of cell signaling proteins can also help determine a cytokine""s role in physiology. For example, signaling involving signal transducers and activators of transcription-4 (STAT4) may indicate an inflammatory response, while signaling via STAT6 may indicate an anti-inflammatory response or Th2-type response (Romagnani (1997) Immunol. Today 18:263-266). Hence, the identities of the proteins that are phosphorylated, of the particular amino acid residues that become phosphorylated, and of the relevant transcription factors and genes that become activated, are all relevant to determining the function of the cytokine of interest. Other powerful techniques for determining the functions of cytokines and related signaling proteins include use of genetically altered animals where a specific gene is altered in all cells, e.g., IL-10xe2x88x92/xe2x88x92 mice (Scheerens, et al., (2001) Eur. J. Immunol. 31:1465-1474), or where the genetic alteration is targeted to only one type of cell in the animal, e.g., liver cells, epithelial cells, cells of lymphoid lineage (Blumberg, et al., (2001) Cell 104:9-19), or macrophages and neutrophils (Takeda, et al. (1999) Immunity 10:39-49).
The present invention is directed to mammalian, e.g., rodent, canine, feline, primate, AK155 (interleukin-XX; IL-XX) and its biological activities. It includes nucleic acids coding for polypeptides themselves and methods for their production and use. The nucleic acids of the invention are characterized, in part, by their homology to cloned complementary DNA (cDNA) sequences enclosed herein, or by functional assays for IL-10-like activities of the polypeptides encoded by these nucleic acids. Methods for modulating, inhibiting, or stimulating an immune response are provided.
The present invention is based, in part, upon the discovery of a new cytokine exhibiting a sequence similarity to cellular IL-10. In particular, it provides a gene encoding a protein whose mature size is about 150 amino acids, which is expressed in virally transformed cells, and in certain tissues, e.g., kidney, and possibly lung and liver. Functional equivalents exhibiting significant sequence homology will be available from various mammalian species, i.e., human, mouse, rat.
More particularly, the present invention provides a substantially pure or recombinant AK155 protein or peptide fragment thereof. Various embodiments include an antigenic protein or peptide selected from a protein or peptide from a warm blooded animal selected from the group of birds and mammals, including a primate; a protein or peptide comprising at least one polypeptide segment of SEQ ID NO:2 or a fragment thereof; a protein or peptide which exhibits a post-translational modification pattern distinct from natural AK155; or a protein or peptide which is capable of co-stimulating a T cell with another signal. The protein or peptide can comprise a fusion protein. Another embodiment is a composition comprising an AK155 protein or peptide and a pharmaceutically acceptable carrier.
The invention also embraces an antibody which specifically binds a AK155 protein or peptide, e.g., wherein the AK155 is a mammalian protein, including a primate; the antibody is raised against a purified AK155 peptide sequence of SEQ ID NO:2; the antibody is a monoclonal antibody; or the antibody is labeled. The antibodies also make available a method of purifying an AK155 protein or peptide from other materials in a mixture comprising contacting the mixture to an anti-AK155 antibody, and separating bound AK155 from other materials.
Another aspect of the invention is an isolated or recombinant nucleic acid capable of encoding a full length or mature AK155 polypeptide, including a nucleic acid which encodes a sequence of SEQ ID NO:2; which includes a sequence of SEQ ID NO:1; or which encodes a sequence from a natural AK155. Such nucleic acid embodiments also include an expression vector.
The invention also provides a kit containing a substantially pure AK155 or fragment; an antibody or receptor which specifically binds AK155; or a nucleic acid, or its complement, encoding AK155 or a fragment thereof. This kit also provides methods for detecting in a sample the presence of a nucleic acid, protein, or antibody, comprising testing said sample with such a kit.
The invention also supplies methods of modulating the physiology of a cell comprising contacting said cell with a substantially pure AK155 or fragment; an antibody or binding partner which specifically binds an AK155; or a nucleic acid encoding an AK155 or peptide. Certain preferred embodiments include a method where the cell is a T cell and the modulating of physiology is activation of the T cell or apoptosis of the T cell; or where the cell is in a tissue or in an organism.
Also provided are methods of expressing an AK155 peptide by expressing a nucleic acid encoding an AK155 polypeptide. The invention also provides a cell, tissue, organ, or organism comprising a nucleic acid encoding an AK155 peptide.
The invention also provides a recombinant nucleic acid comprising sequence at least about 70% identity over a stretch of at least about 30 nucleotides to an AK155 nucleic acid sequence of SEQ ID NO:1, useful, e.g., as a probe or PCR primer for a related gene. Another embodiment further encodes a polypeptide comprising at least about 60% identity over a stretch of at least about 20 amino acids to an AK155 sequence of SEQ ID NO:2.
The invention further provides a method of treating a patient having an immune or inflammatory response by administering an effective dose of an antibody or binding partner specific for AK155; an AK155 protein or polypeptide; or a nucleic acid encoding an AK155 peptide.
xe2x80x9cAK155xe2x80x9d refers to a polypeptide having a sequence that has greater than 70% amino acid sequence identity, preferably greater than 75%, 80%, 85%, 90%, or 95% amino acid sequence identity, to SEQ ID NO:2.
xe2x80x9cAK155 receptorxe2x80x9d refers to a polypeptide complex that is a comprised of an xcex1-subunit and xcex2-subunit. The term AK155 receptor therefore refers to polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have the characteristic that they are activated by the binding of AK155; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of the xcex1-subunit or xcex2-subunit of the AK155 receptor.
xe2x80x9cCytokinesxe2x80x9d are proteins that regulate and coordinate many of the activities of the immune system. Cytokines produced by mononuclear phagocytes have been called monokines, while those produced by lymphocytes have been called lymphokines. Most of the cytokines have numbered names while others have trivial names, such as interferon (IFN) and tumor necrosis factor (TNF) (Abbas, et al. (2000) Cellular and Molecular Immunology, 4th ed., W. B. Saunders Co., New York, N.Y., pp. 235, 486).
xe2x80x9cIL-10-related cytokinesxe2x80x9d refers to cytokines with homology to IL-10. These cytokines include IL-20, IL-22, IL-19, melanoma differentiation-associated gene 7 (mda-7), and AK155 (Dumoutier, et al. (2001) J. Immunol. 167:3545-3549). The IL-10-related cytokines also include the IL-10 homologues of Epstein-Barr virus, equine herpesvirus type 2, and parapoxyirus (Knappe, et al. (2000) J. Virology 74:3881-3887).
xe2x80x9cRecombinantxe2x80x9d when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of an exogenous, non-native nucleic acid or the alteration of a native nucleic acid, or that the cell is derived from a cell so modified.
xe2x80x9cExogenousxe2x80x9d refers to substances that are produced outside an organism or cell, depending on the context.
xe2x80x9cAnti-inflammatoryxe2x80x9d refers to the reduction of inflammation, and includes reducing a local or systemic response to cellular injury that is marked by capillary dilation and leukocytic infilatration out of the capillaries and into the surrounding tissues. xe2x80x9cAnti-inflammatoryxe2x80x9d also refers to the reduction of any of the classic signs of inflammation, i.e., rubor (redness), tumor (swelling), calor (heat), and dolor (pain).
xe2x80x9cFunctional effectsxe2x80x9d in the context of assays for testing compounds affecting a receptor comprising an AK155 receptor includes the determination of any parameter that is indirectly or directly under the influence of the receptor. It includes changes in the conformation of an AK155 receptor, changes in the strength or nature of association and binding of various proteins, cofactors, and ligands to an AK155 receptor, changes in the percentage of AK155 receptor that is cytosolic or membrane-bound, changes in amount of surface expression of AK155 receptor or in the amount of surface-association of proteins that bind to an AK155 receptor, changes in protein phosphorylation, in the rate of transcription of a gene, in the rate of secretion, in the rate of cell proliferation, and in cell maturation or differentiation.
xe2x80x9cInhibitorsxe2x80x9d and xe2x80x9cactivatorsxe2x80x9d of the AK155 receptor refer to inhibitory or activating molecules, respectively, identified using in vitro and in vivo assays for AK155 receptor activation. A xe2x80x9cmodulatorxe2x80x9d of AK155 receptor activation is a molecule that is an inhibitor or an activator of AK155 receptor. Inhibitors are compounds that decrease, block, prevent, delay activation, inactivate, desensitize, or down regulate the receptor. Activators are compounds that increase, activate, facilitate, enhance activation, sensitize or up regulate receptor activity. To examine the extent of inhibition, samples or assays comprising an AK155 receptor are treated with a potential activator or inhibitor and are compared to control samples without the inhibitor. Control samples (untreated with inhibitors) are assigned a relative AK155 receptor activity value of 100%. Inhibition of the AK155 receptor is achieved when the AK155 receptor activity value relative to the control is about 90%, preferably 50%, more preferably 5-25%. Activation of the AK155 receptor is achieved when the AK155 receptor activity value relative to the control is 110%, more preferably 150%, still more preferably at least 2-fold higher, and most preferrably at least 5-fold higher.
xe2x80x9cCell linexe2x80x9d refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. Spontaneous or induced changes can occur in the genome can occur during storage or transfer of one or more cells present in the population of cells. Therefore, cells derived from a cell line may not be precisely identical to the ancestral cells or cultures, and the cell line includes such variants. The term xe2x80x9ccell linesxe2x80x9d also includes immortalized cells (U.S. Pat. No. 6,090,611 issued to Covacci, et al.).
By xe2x80x9cpurifiedxe2x80x9d and xe2x80x9cisolatedxe2x80x9d is meant, when referring to a polypeptide, that the polypeptide is present in the substantial absence of the other biological macromolecules of the same type. The term xe2x80x9cpurifiedxe2x80x9d as used herein preferably means at least 85%, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type present. The weights of water, buffers, salts, detergents, reductants, protease inhibitors, stabilizers, excipients, and other small molecules, especially those having a molecular weight of less than 1000, are not used in the determination of polypeptide purity (U.S. Pat. No. 6,090,611 issued to Covacci, et al.). Purity and homogeneity are typically determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), optionally with analysis using a gel scanner (Brody, T. (1997) Analyt. Biochem. 247:247-256), high pressure liquid chromatography (HPLC) or capillary electrophoresis (CE) (Gooding and Regnier (2002) HPLC of Biological Molecules, 2nd ed., Marcel Dekker, NY; Cunico, Gooding, and Wehr (1998) Basic HPLC and CE of Biomolecules, Bay Biological Laboratory, Inc. Hercules, Calif.), and N-terminal (Abe, et al. (1998) J. Biol. Chem. 273:11150-11157) or C-terminal amino acid sequencing (Boyd, et al. (1992) Analyt. Biochem. 206:344-352). A protein that is the predominant species present in a preparation is substantially purified. It is understood that 100% purity may be an impossibility, e.g., because of low levels of trace amounts of proteases or natural deamidation (Hsu, et al. (1998) Biochemistry 37:2251-2262; Robinson and Robinson (2001) Proc. Natl. Acad. Sci. USA 98:12409-12413; Sarioglu, et al. (2000) Electrophoresis 21:2209-2218).
xe2x80x9cNucleic acidxe2x80x9d refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single stranded or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids.
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., (1991) Nucleic Acids Res. 19:5081; Ohtsuka, et al. (1985) J. Biol. Chem. 260:2605-2608; Rossolini et al. (1994) Mol. Cell. Probes 8:91-98). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
A particular nucleic acid sequence also implicitly encompasses xe2x80x9callelic variantsxe2x80x9d and xe2x80x9csplice variants.xe2x80x9d Similarly, a particular protein encoded by a nucleic acid implicitly encompasses any protein encoded by an allelic variant and splice variant of that nucleic acid. xe2x80x9cSplice variantsxe2x80x9d are products of alternative splicing of a mRNA. After transcription, an initial mRNA may be spliced such that different (alternate) splice products encode different polypeptides. Mechanisms for the production of splice variants vary, but include alternate splicing of exons. Alternate polypeptides derived from the same nucleic acid by read-through transcription are also encompassed by this definition. Any products of a splicing reaction, including recombinant forms of the splice products, are included in this definition. It will be understood that inasmuch as natural allelic variations exist and occur from individual to individual, as demonstrated by amino acid differences in the overall sequence or by deletions, substitutions, insertions, inversions, or additions of one or more amino acids of said sequences, the present invention is intended to embrace all of such allelic variations of the two molecules involved. In addition, the location of and the degree of glycosylation depend upon the nature of the recombinant host organism employed as well as disease, i.e., rheumatic disease and rheumatoid arthritis, and such variations as may occur are included within the ambit of this invention (Jefferis (2001) BioPharm 14:19-27; Mimura, et al. (2001) J. Biol. Chem. 276:45539-45547; Axford (1999) Biochim. Biophys. Acta 1:219-229; Malhotra, et al. (1995) Nature Med. 1:237-243).
xe2x80x9cAmino acidxe2x80x9d refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, including selenomethionine, as well as those amino acids that are modified after incorporation into a polypeptide, e.g., hydroxyproline, xcex3-carboxyglutamate, O-phosphoserine, and cystine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an xcex1-carbon that is bound by a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by their one-letter symbols.
xe2x80x9cConservatively modified variantsxe2x80x9d applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are xe2x80x9csilent variations,xe2x80x9d which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a conserved amino acid or a small percentage of amino acids in the encoded sequence is a xe2x80x9cconservatively modified variant.xe2x80x9d Conservative substitution tables providing functionally similar amino acids are well known in the art. An example of a conservative subtitution is the exchange of an amino acid in one of the following groups for another amino acid of the same group (U.S. Pat. No. 5,767,063 issued to Lee, et al.; Kyte and Doolittle (1982) J. Mol. Biol. 157:105-132):
(1) Hydrophobic: Norleucine, lie, Val, Leu, Phe; Cys; or Met;
(2) Neutral hydrophilic: Cys, Ser, Thr;
(3) Acidic: Asp, Glu;
(4) Basic: Asn, Gin, His, Lys, Arg;
(5) Residues that influence chain orientation: Gly; Pro.
(6) Aromatic: Trp; Tyr; Phe.
(7) Small amino acids: Gly, Ala, Ser.
For example, when no change or an insignificant change in biological function is desired, substitutions can be made by changing Val to Leu; Ser to Thr; or Asp to Glu.
Polypeptide molecules having substantially the same amino acid sequence as AK155 but possessing minor amino acid substitutions that do not substantially affect the functional aspects are within the definition of the contemplated invention. Variants with truncations or deletions of regions which do not change the biological functions of AK155 are also within the definition of the contemplated invention. Variants containing one or more peptide bond cleavages, where daughter polypeptides remain in association with each other, are within the definition of the contemplated invention. Where a polypeptide chain is cleaved, e.g., during normal processing or because of damage due to non-specific protease action, daughter polypeptides can maintain their association with each other because of covalent linkage via a disulfide bond or because of three dimensional conformations that allow or maintain a plurality of ionic bonds between the daughter polypeptides. Cleaved variants are contemplated, e.g., where cleavage results in a maintenance of antigenic activity or signal-transducing activity.
The term xe2x80x9cproteinxe2x80x9d generally refers to the primary sequence of amino acids forming the polypeptide chain, any post-translational modifications of the proteins, dimers, and multimers of the polypeptide chain, and the three dimensional structure of the polypeptide. xe2x80x9cDenatured proteinxe2x80x9d refers to a partially or totally denatured polypeptide, having some residual three dimensional structure or, alternatively, an essentially random three dimensional structure. When applied to a specific polypeptide, the term xe2x80x9cproteinxe2x80x9d may or may not include covalent or non-covalent modifications, i.e., salts or cofactors associated with the protein, depending on the circumstances. For example, when a term refers to an aminotransferase, the term generally includes the non-covalently bound cofactor that is bound to the aminotransferase.
A xe2x80x9cpromoterxe2x80x9d is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A xe2x80x9cconstitutivexe2x80x9d promoter is a promoter that is active under most environmental and developmental conditions, while an xe2x80x9cinduciblexe2x80x9d promoter is a promoter that is active under environmental or developmental regulation.
The term xe2x80x9coperably linkedxe2x80x9d refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
An xe2x80x9cexpression vectorxe2x80x9d is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular open reading frame in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment. Typically, the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
The terms xe2x80x9cidenticalxe2x80x9d or percent xe2x80x9cidentity,xe2x80x9d in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same i.e., 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identity over a specified region when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition also refers to the complement of a polynucleotide sequence.
Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches, if necessary, by introducing gaps as required. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Sequence Comparison, Addison-Wesley, Reading, Mass.; and software packages, such as MacVector 7.1(copyright) from Accelrys, Inc. (San Diego, Calif.) and Vector NTI(copyright) Suite from InforMax, Inc. (Bethesda, Md.). xe2x80x9cSequence identityxe2x80x9d means that in comparing two polypeptides, the identities of all of the corresponding amino acids in that stretch are one and the same, i.e., where there is a histidine in polypeptide A there is a histidine in polypeptide B. In determining a value for sequence identity, the amino acids of N-terminal or C-terminal extensions are ignored. xe2x80x9cSequence similarityxe2x80x9d means that in comparing two polypeptides, the identities of all of the corresponding amino acids are one and the same, or correspond to a conservative substitution. Conservative substitutions are defined above in Definitions. Typical homologous proteins or peptides will have from 25-100% homology. Homology measures will be at least about 20%, generally at least about 30%, often at least about 40%, typically at least about 50%, usually at least about 60%, preferably at least about 70%, and more preferably at least about 80%.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. For sequence comparison of nucleic acids and proteins to AK155 receptor nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.
A xe2x80x9ccomparison windowxe2x80x9d, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms, as those provided by software packages, such as MacVector 7.1(copyright) from Accelrys, Inc. (San Diego, Calif.) and Vector NTI(copyright) Suite from InforMax, Inc. (Bethesda, Md.).
A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul, et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul, et al. (1990) J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information, on the world wide web at xe2x80x9cncbi.nlm.nih.gov.xe2x80x9d This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul, et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always greater than 0) and N (penalty score for mismatching residues; always  less than 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=xe2x88x924 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff(1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=xe2x88x924, and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
An indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions. Where the two polypeptides are substantially similar, 25% of the positions of non-identity are due to conservative changes, where more preferably 50% of the positions of non-identity are due to conservative changes, and still more preferably 90% of the positions of non-identity are due to conservative changes.
The phrase xe2x80x9cselectively (or specifically) hybridizes toxe2x80x9d refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
The phrase xe2x80x9cstringent hybridization conditionsxe2x80x9d refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. A guide to the hybridization of nucleic acids is available (Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biologyxe2x80x94Hybridization with Nucleic Probes, Vol. 24, Parts 1 and 2, Elsevier, New York, N.Y.). Generally, stringent conditions are selected to be about 5-10xc2x0 C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30xc2x0 C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60xc2x0 C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For high stringency hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5xc3x97SSC and 1% SDS incubated at 42xc2x0 C. or 5xc3x97SSC and 1% SDS incubated at 65xc2x0 C., with a wash in 0.2xc3x97SSC and 0.1% SDS at 65xc2x0 C.
Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary xe2x80x9cmoderately stringent hybridization conditionsxe2x80x9d include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37xc2x0 C., and a wash in 1xc3x97SSC at 45xc2x0 C. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency.
xe2x80x9cAntibodyxe2x80x9d refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one xe2x80x9clightxe2x80x9d (about 25 kD) and one xe2x80x9cheavyxe2x80x9d chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (VL) and variable heavy chain (VH) refer to these light and heavy chains respectively.
Antibodies occur as intact immunoglobulins, as fragments produced by digestion with various peptidases, or as recombinant varieties, such as humanized antibodies or single chain antibodies. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)xe2x80x22, a dimer of Fab which itself is a light chain joined to VH-CH1 by a disulfide bond. The F(ab)xe2x80x22 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)xe2x80x22 dimer into an Fabxe2x80x2 monomer. The Fabxe2x80x2 monomer is essentially Fab with part of the hinge region.
In an antibody fragment comprising one or more heavy chains, the heavy chain(s) can contain any constant domain sequence (e.g. CH1 in the IgG isotype) found in a non-Fc region of an intact antibody, and/or can contain any hinge region sequence found in an intact antibody, and/or can contain a leucine zipper sequence fused to or situated in the hinge region sequence or the constant domain sequence of the heavy chain(s). Suitable leucine zipper sequences include the jun and fos leucine zippers and the GCN4 leucine zipper (Kostelney, et al. (1992) J. Immunol. 148:1547-1553; U.S. Pat. No. 6,133,426 issued to Gonzalez, et al.).
While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant methodologies, such as recombinant IgG antibodies (U.S. Pat. No. 4,816,567 issued to Cabilly, et al; U.S. Pat. No. 4,642,334; issued to Moore, et al.; Queen, et al. (1989) Proc. Natl Acad. Sci. USA 86:10029-10033), single chain antibodies, or antibodies acquired by phage display, and monoclonal antibodies made by the hybridoma method (Kohler, et al. (1975) Nature 256:495).
The synthesis of single chain antibodies is described in U.S. Pat. No. 4,946,778 issued to Ladner, et al., while single domain antibodies are described by Conrath, et al. (2001) J. Biol. Chem. 276:7346-7350, and Desmyter, et al. (2001) J. Biol. Chem. 276:26285-26290). Antibodies may also be produced by the phage display technique (Barbas, et al. (2001) Phage Display: A LaboratorY Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Kay, et al. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, Calif.; de Haard, et al. (1999) J. Biol. Chem. 274:18218-18230; McCafferty et al. (1990) Nature 348:552-554; Clackson et al. (1991) Nature 352:624-628; Marks et al. (1991) J. Mol. Biol. 222:581-597).
For preparation of monoclonal or polyclonal antibodies, any technique known in the art can be used (see, e.g., Kohler and Milstein (1975) Nature 256:495-497; Kozbor et al. (1983) Immunology Today 4:72; Cole et al. (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., New York, N.Y., pp. 77-96). Techniques for the production of single chain antibodies (U.S. Pat. No. 4,946,778 issued to Ladner, et al.) can be adapted to produce antibodies to polypeptides of this invention. Also, transgenic mice, or other organisms such as other mammals, may be used to express humanized antibodies.
An xe2x80x9canti-AK155 receptor antibodyxe2x80x9d is an antibody or antibody fragment that specifically binds an AK155 receptor or subunits thereof.
A xe2x80x9cchimeric antibodyxe2x80x9d is an antibody molecule in which part or all of the constant region is altered, with a replacement or exchange, so that the antigen binding site is linked to a constant region of a different class or antibody, or to an enzyme, hormone, protein toxin (U.S. Pat. No. 6,051,405 issued to Fitzgerald, et al.), growth factor, or drug.
An xe2x80x9cimmunoassayxe2x80x9d is an assay that uses an antibody to specifically bind an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, or quantify the antigen.
xe2x80x9cBiological samplexe2x80x9d as used herein is a sample of biological tissue or fluid that contains the AK155 receptor or nucleic acid encoding the subunits of the AK155 receptor proteins. Such samples include, but are not limited to, tissue isolated from humans. Biological samples may also include sections of tissues such as frozen sections taken for histologic purposes. A biological sample is typically obtained from a eukaryotic organism, preferably eukaryotes such as fungi, plants, insects, protozoa, birds, fish, reptiles, and preferably a mammal such as rat, mice, cow, dog, guinea pig, or rabbit, and most preferably a primate such as chimpanzees or humans.
The phrase xe2x80x9cspecificallyxe2x80x9d or xe2x80x9cselectivelyxe2x80x9d binds, when referring to a ligand/receptor or other binding pair, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologics. Thus, under designated conditions, a specified ligand binds to a particular receptor and does not bind in a significant amount to other proteins present in the sample.
In a preferred embodiment of the invention, AK155 or AK155 variant or mutein binds to an AK155 receptor or AK155 receptor subunit with an affinity that is ten times greater, more preferably 20-times greater, still more preferably 40-times, and most preferably 80-times greater than the affinity found when tested with any other proteins, aside from anti-AK155 antibodies.
In a preferred embodiment of the invention, anti-AK155 antibody binds to AK155, or a variant or mutein thereof, with an affinity that is ten times greater, more preferably 20-times greater, and still more preferrably 40-times greater, and most preferrably 80-times greater than the affinity with any other antibody. In a preferred embodiment of the monoclonal antibody to AK155, or to a variant or mutein thereof, the monoclonal antibody will have an affinity which is greater than about 109 liters/mol, and preferably is equal to or greater than about 1010 liters/mol, as determined, for example, by Scatchard analysis (Munsen, et al (1980) Analyt. Biochem. 107:220-239). Further information on antibody affinity constants is available (Friguet, et al., (1985) J. Immunol. Methods 77:305; Hubble (1997) Immunol. Today 18:305-306).
xe2x80x9cLigandxe2x80x9d refers to an entity that binds specifically to a polypeptide or a complex of more than one polypeptide. A xe2x80x9cligand binding domainxe2x80x9d is a polypeptide or region of a polypeptide that is able to bind to said entity. A ligand may be a soluble protein, a membrane-associated protein, or an integral membrane-bound protein. Where a ligand binds to a receptor, the question of which molecule is the ligand and which molecule is the receptor can be determined on a case-by-case basis. Generally, where the binding event results in cell signaling, the entity (of the two entities being discussed) that is involved in downstream signaling is termed the xe2x80x9creceptor.xe2x80x9d A freely diffusable and water-soluble entity that is involved in ligand/receptor interactions is usually a ligand, not a receptor.
An xe2x80x9cagonistxe2x80x9d is a compound that interacts with a target and causes an increase in the activation of the target.
An xe2x80x9cantagonistxe2x80x9d is a compound that opposes the actions of an agonist. An agonist prevents, inhibits, or neutralizes the activity of an agonist. An antagonist is also a compound that causes a decrease in activity of a target.
The term xe2x80x9cmonoclonal antibodyxe2x80x9d (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier xe2x80x9cmonoclonalxe2x80x9d indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
xe2x80x9cMonoclonal antibodiesxe2x80x9d also include clones of antigen-recognition and binding-site containing antibody fragments (Fv clones), i.e., isolated from phage antibody libraries.
As used herein a xe2x80x9cnucleic acid probe or oligonucleotidexe2x80x9d is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
A xe2x80x9clabeled nucleic acid probe or oligonucleotidexe2x80x9d is one that is modified, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe. The probes are preferably directly labeled as with isotopes, chromophores, fluorophores, chromogens, or indirectly labeled such as with biotin to which a streptavidin or avidin complex may later bind.
A composition is xe2x80x9clabeledxe2x80x9d that is detectable, either directly or indirectly, by spectroscopic, photochemical, biochemical, immunochemical, radiometric, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes and their substrates (e.g., as commonly used in enzyme-linked immunoassays, e.g., alkaline phosphatase and horse radish peroxidase), biotin-streptavidin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available. The label or detectable moiety is typically bound, either covalently, through a linker or chemical bound, or through ionic, van der Waals or hydrogen bonds to the molecule to be detected.
The term xe2x80x9cradiolabeledxe2x80x9d refers to a compound to which a radioisotope has been attached through covalent or non-covalent means. Examples of radioisotopes include, without limitation, 3H, 14C, 32P, 33P, 35S, and 125I.
A xe2x80x9cfluorophorexe2x80x9d is a compound or moiety that accepts radiant energy of one wavelength and emits radiant energy of a second wavelength.